ES2708699T3 - Determination of the intact mass of protein-conjugated compounds - Google Patents

Abstract

A method for the detection or determination of a mass of an antibody conjugate compound and a drug, comprising: (a) providing a conjugate compound of an antibody with a drug in a volatile salt and free of a non-salt volatile, wherein the conjugate compound of an antibody with a drug has one to eight drug residues conjugated in the reduced intercatenary disulfides, is associated non-covalently and non-denatured, and keeps its bivalent antibody structure intact. (b) introducing the conjugate compound of an antibody with a drug of step a) into a mass spectrometer; and subsequently (c) directly establish the mass of the intact bivalent structure of the conjugate compound of an antibody with a drug by mass spectrometry.

Description

DESCRIPTION

Determination of the intact mass of protein-conjugated compound compounds

Background of the invention

Drug-antibody conjugates (ADCs) are compounds for the targeted delivery of their target load. In many cases, the target is an antigen associated with a tumor (TAA) and the payload is a drug.

There are several classes of antibodies that include IgG1 and IgG2. The structure of an IgG1 and IgG2 antibody includes two heavy chains (HC) and 2 light chains (LC). The two heavy chains and the two light chains are presented as a complex comprising an intact antibody. Each IgG1 antibody has 12 intrachain disulfide bonds and four interchain linkages created by the oxidation of their respective cysteines in each antibody chain. There are two disulfide bonds between the heavy chain and the light chain and two disulfide bonds between the heavy chain and heavy chain. The complete reduction of the interchain disulfide bonds of an IgG1 antibody results in the antibody complex being maintained by non-covalent interactions.

The complete reduction of the intrachain disulfide bonds of an IgG1 antibody gives rise to eight accessible thiols for conjugation with a drug, generally by means of a linker. By varying the reduction parameters, antibody isomers can be obtained with from 0 to 8 thiols available for conjugation. For example, by reducing an IgG1 with DTT, antibodies can be obtained with accessible 2, 4, 6, or 8 thiols. Fully reduced DNAs are held together by non-covalent interactions, such as hydrogen bonds, ionic bonds, hydrophobicity and van der Waals interactions, and separated into light and heavy chains under denaturing conditions (eg, reversed-phase chromatography). ). The unreduced ADCs are completely held together by covalent and non-covalent interactions. Parts of fully reduced ADC that are held together by covalent media can also be separated under denaturing conditions.

The fully loaded IgG1 ADCs have a drug molecule bound to each cysteine that produces the interchain disulfides of an antibody with a total of eight drugs per antibody. The partially charged ^c D generally have 2, 4, or 6 drug molecules bound residues cistelna. Partially charged ADCs having 1, 3, and 5 drug molecules attached to the cysteine residues are also observed. Although the mass of the constituent fragments of the charged ADCs has been tested by mass spectrometry (MS), there remains a problem in the field to which the present invention relates with respect to the assay of the mass of the charged intact ADC.

Although techniques for direct measurement of the mass of an intact charged ADC are not available, various indirect means are known for measuring the mass of the charged ADCs. For example, the mass of an ADC has been measured by joining a sample (eg, an ADC) to a high pressure liquid chromatographic column. Reverse phase heated (rp-HPLC) in the presence of little or no organic solvent also normally containing an ionic acid pairing (eg, trifluoroacetic acid) and allowing the non-volatile salts and surfactants of the sample to be washed ( which is commonly referred to as desalination). In this example, the protein is eluted from the rp-HPLC column by increasing the organic content of the solvent to the point where the interactions between the hydrophobic protein domains and the surface of the rp-HPLC column are destroyed by an organic solvent not polar. An undesirable effect of this technique is that the protein structure is destroyed by subjecting the protein samples to heat, acid and organic solvents, any of which can denature the proteins and destroy the protein structure. When the non-covalent protein complexes are subjected to a rp-HPLC they are separated into their covalent entities that constitute them. In the case of an IgG1 antibody with 8 drugs bound to the interchain cysteines, the desalting in a column of rp-HPLC results in the complete dissociation of the ADC in the heavy chains with 3 drugs per chain and the light chains with 1 drug per chain. Although the mass of the constituent fragments can be determined by mass spectrometric (MS) techniques, the techniques for determining the masses of the intact entity do not exist in the field to which the present invention pertains.

Current MS techniques lack the measurement of intact ADC mass due, in part, to the fact that these techniques result in protein denaturation and / or consume too much time for the uses contemplated in this document. Virtually all methods of MS with ionization and native electrospray (ESI) of the proteins specify that this procedure should be carried out at the nanospray scale (with a flow rate of 100 to 500 nanoliters / minute) to minimize the destruction of the protein structure. that will be produced by the heated cover and the desolvation of gases that are used for the conventional ESI. The procedure of handling the sample for the measurement of the native mass of an ADC using MS-ESI techniques with conventional nanospray is time consuming and not suitable for high performance. Not including the time to deglycosylate the antibody, it takes at least one hour per sample to obtain a mass measurement.

Lazar et al., Rapid Communications in Mass Spectrometry 2005,19 (13): 1806-1814 discloses an MS-ESI for the analysis of recombinant and immunoconjugated antibodies, including sample preparation and desalting of the sample. Wakankar et al., MABS 2011, 3 (2): 161-172 reviews the analytical methods used for the characterization of ADCs, such as protein mass spectrometry and capillary electrophoresis.

In addition, previously known methods for analyzing the ADCs bound to the intercatenary cysteinyls are also unsuitable for line mass spectrometry (eg, HIC) or result in the denaturing dissociation of the heavy and light chains conjugated during the chromatographic separation and the subsequent measurement of mass (for example, rp-HPLC). Therefore, there is a need in the field to which the present invention pertains for methods for the routine and rapid determination of the intact mass of a DC coupled to cysteines.

Surprisingly, the present invention fulfills this need so! as other needs not met in the field by providing methods, devices, and systems for detecting the mass of a non-covalently associated protein agent conjugate.

Brief summary of the invention

In one aspect, the invention provides a method for the detection or determination of a mass of drug antibody conjugated compound. The method includes the steps of providing a drug antibody conjugate compound in a volatile salt and hare of a non-volatile salt, wherein the drug antibody conjugate compound has from one to eight drug moieties conjugated to the reduced interchain disulfides, associates non-covalently and is not denatured, and maintains its bivalent antibody structure intact; the introduction of the drug antibody conjugate compound into a mass spectrometer; and directly establishing the mass of the intact bivalent structure of the drug antibody conjugate compound by mass spectrometry. Brief description of the drawings

Figure 1 shows the IgG1 structures of various representative ADCs with an MR of 0 to 8 including the isomers produced by conjugation of an antibody with an agent that is, in one aspect, a drug, in another aspect, a marker, and in another aspect, a toxin. The disulfide bonds between the antibody subunits (referred to as mAb in the Figure) (MR = 0) are bound to drugs in the ADC that result in non-covalently associated 2LC-2HC drug bound species.

Figure 2 shows the unprocessed or disaggregated MS associated with the measurement of the deglycosylated mAb-A mass under denaturing and non-denaturing conditions. The unprocessed and non-disaggregated MS data obtained under the denaturing conditions are shown in panels A and C, respectively, and the unprocessed and disaggregated MS data obtained under non-denaturing conditions are shown in panels B and D, respectively. The apparent ions between 200 and 3500 m / z of panel B which is the region in which the denatured antibody will be evident are due to the presence of PNGase F which was used for deglycosylation and the non-ionic detergent Tween-80.

Figure 3 shows the unprocessed and disaggregated MS associated with the mass measurement of a deglycosylated maleimidocaproyl monomethyl auristatin F (mcMMAF), ADC-A conjugate under denaturing and non-denaturing conditions. The unprocessed and disaggregated MS data obtained under denaturing conditions are shown in panels A and C, respectively, and unprocessed and disaggregated MAS data obtained under non-denaturing conditions are shown in panels B and D, respectively. The multiple ions charged for the ADC-A are indicated by brackets in panel B and the breakdown artifacts present in panels C and D are indicated by asterisks.

Figure 4 shows a disaggregated mass spectrum for a deglycosylated maleimidocaproyl monomethyl auristatin F (mcMMAF) conjugate ADC-A and the corresponding parent material, mAb-A. The non-covalently associated ADC structures are shown above the corresponding ion in the MS.

Figure 5 shows a disaggregated mass spectrum for a deglycosylated ADC-B conjugated monomethyl auristatin D (vcMMAE) mc-Val-Cit-PAB conjugate E and the corresponding parent material, mAb-B. Non-covalently associated ADC structures are shown on top of the corresponding ion in the MS.

Figure 6 shows the relative levels of the molar ratios of vcMMAF by IgG1 ADC-A (panel A) and vcMMAE by IgG1 ADC-B (panel B), as determined by MS-based quantification for the mass spectra and by UV integration of the species separated by BIC.

Figure 7 shows a comparison of the chromatograms of the dual column SEC analyzes of the desalted ADC and the corresponding starting material.

Figure 8 shows the relative percentage of vcMMAE by concentration of salts for the conjugates having MR value of 0, 2, 4, 6, and 8.

Figure 9 results of a separation characterization of a mcMMAF DNA by HIC (panel A), followed by an analysis of the fractions collected by SEC-MS (panel B).

Figure 10 shows an analysis of the individual fractions of the HIC separation showing that the position of the drug binding in the polypeptide chains of the ADC can be assessed by the mass of the dissociated fragments using SEC-MS.

Figure 11 shows a mass measurement by MS broken down for a protein conjugate with an agent having an MR = 0, 2, 4, 6, or 8.

Figure 12 shows a mass measurement by MS broken down for a protein conjugate with an agent having an MR = 0, 2, 4 or 6.

Detailed description of the invention

I. General

The present disclosure discloses methods for detecting the mass of a protein conjugate compound with a non-covalently associated agent. These methods include the steps of: (a) providing a protein conjugate compound with an agent in a matrix; (b) introducing the protein conjugate compound with an eluted agent in a mass spectrometer; and (c) directly establishing the mass of the protein conjugate compound with an agent by mass spectrometry. In further embodiments, a separation medium is applied under non-denaturing conditions for the protein conjugate compound with a matrix agent to effect the separation of the protein agent conjugate compound from the matrix. In some of these embodiments, the protein conjugate compound with an agent is substantially non-denatured. In still further embodiments, the protein conjugate compound with a non-denatured agent is eluted from the separation medium compound in a volatile salt.

II. Definitions

Unless otherwise defined, all terms of the art, notations and other scientific terms or terminology used herein are meant to have the meanings commonly understood by those skilled in the art to which the present invention pertains. . In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions in this document will not necessarily interpret it as representing a substantial difference over what is understood in general in the technique. Many of these techniques and methods described or referenced herein are well understood and commonly employed using a conventional methodology by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with protocols and / or parameters defined by the manufacturer unless otherwise noted.

When trade names are used herein, it is intended that the product formulation of the trade name, the genetic drug, and the active pharmaceutical ingredients of the product of commercial name be independently included.

As used herein, the abbreviation "MR" refers to the number of drug molecules conjugated with, for example, the protein or the antibody. For example, MR = 0 means that zero drug molecules have been conjugated to a given protein or a given antibody. MR = 8 means that there are eight drug molecules conjugated with a given protein or a given antibody. MR can include 0, 1, 2, 3, 4, 5, 6, 7 and 8.

Unless otherwise stated, the following terms and phrases as used in this document are intended to have the following meanings:

As used herein, the term "ADC" refers to an antibody drug conjugate. The ADC antibody portion has specificity for an antigen. Antigens of interest include but are not limited to CD30, CD40, CD19, CD33, and CD70.

As used herein, the term "protein" refers to compounds that comprise one or more polypeptides normally folded in a three-dimensional form, and which may provide a biological function.

As used herein, the term "polypeptide" refers to an amino acid polymer and its equivalent and does not refer to a specific product length; therefore "peptides" and "proteins" are included in the definition of polypeptide. It is also included in the definition of "antibodies" as defined in this document. A "polypeptide region" refers to a segment of a polypeptide, which segment may contain, for example, one or more domains or motifs (eg, a polypeptide region of an antibody may contain, for example, one or more CDRs) .

As used herein, the term "fragment" refers to a portion of a polypeptide that normally has at least 20 contiguous amino acids or at least 50 contiguous amino acids of the polypeptide.

As used herein, the phrase "non-covalently associated protein" refers to a protein in which at least one covalent association between the chains of the polypeptide has been altered and which maintains its tertiary or quaternary structure. For example, in reference to an ADC, a non-covalently associated protein is a protein that undergoes reduction of its interchain disulfide bonds while maintaining its structure intact (ie, the two heavy chains remain associated with the two light chains). . ADCs are composed of covalent and non-covalent interactions. Covalent interactions are maintained in some ADCs, for example, ADCs that have 2, 4, or 6 pharmacological loads. In one example of an ADC having 2 pharmacological charges, the ADC has covalent interchain disulfides between both heavy chains and one between the light chain and the heavy chain with the other light chain with the heavy chain as a non-covalent structure. In the same way, in an example of an ADC loaded with 4 drugs, it is also composed of covalent and non-covalent interactions between the heavy and light chains.

As used herein, the phrase "compound of protein conjugate with an agent" refers to a compound that includes a protein conjugated with an agent. The protein can be, but is not limited to, an antibody, an antibody fragment, a Fc fusion protein, or a non-covalent protein complex. In some aspects the protein can be an antibody. In another aspect, the protein is an antibody fragment. In other respects the protein may be a fusion protein with Fc. In other additional aspects, the protein may be composed of a complex of subunits not linked by covalent bonds. Examples of these types of proteins are hemoglobin, which is a tetrameric set of 2 alpha chains and 2 non-covalently associated beta chains, Concanavalin A which is a tetramer, and the estrogen receptor which is a dimer. The fusion proteins with Fc involve the grafting of a functional protein or a protein domain in a dimeric Fc so that the Fc monomers are bound by cysteine residues in the hinge region of the Fc. The protein may have a quaternary structure that is not held together by covalent bonds but only by non-covalent interactions. An example is hemoglobin that is composed of 4 subunits that can be dissociated under denaturing conditions. Hemoglobin cysteines can be conjugated with drugs, but, unlike antibodies, cysteines do not currently participate in the covalent attachment of the subunit structure.

As used herein, the term "agent" refers to a drug, a label, toxin or the like.

As used herein, the term "matrix" refers to the context or medium in which the protein or conjugate compound of an agent with a protein is present. For example, a "matrix" includes formulation buffers, biological serum, surfactants, excipients, or cell culture media.

As used herein, the phrase "non-denaturing condition" refers to a condition in which proteins (eg, antibodies) do not lose their secondary, tertiary or quaternary structures. Non-denaturing conditions include the absence of heat in excess of the level that would cause a thermal display (e.g., 50 ° C or more) and pH extremes (e.g., below a pH of 5 and above a pH of 8) . As used in the present document, the phrase "non-denatured protein" means a protein (for example, an antibody) that has maintained its secondary, tertiary, and, when applicable, quaternary structure.

As used herein, the phrase "reduced protein" (e.g., an antibody) refers to a protein in which its interchain disulfide bonds have been broken.

As used herein, the phrase "denatured protein" (e.g., an antibody) is in which the protein has lost its secondary, tertiary or quaternary structure.

As used herein, the term "substantially" means a refers to the mobile liquid phase which is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% , or 10% of the amount measured by the relative levels of the molar ratios as determined in the MS-based quantification from the disaggregated mass spectrum and by the UV integration of the species separated by HIC, whose proteins (for example , antibodies) are not denatured following the separation step in the medium (for example, by SEC).

As used herein, the term "eluent" refers to the mobile liquid phase that dissociates analytes from a stationary phase on a chromatography column. Analytes include, but are not limited to, compounds of interest to be measured.

As used herein, the term "directly" refers to the context for the establishment of the mass of a protein (including a conjugate compound of an agent with a protein) that includes the mass measurement of the complete quaternary set , for example, the intact entity, such as an antibody complex.

As used herein, the term "indirectly" means the measurement of the protein mass (including a protein conjugate compound with an agent) by measuring the masses of the subunits comprising the quaternary assembly, for example, the heavy and light chains of an antibody, and summing the masses together to arrive at a number for the intact entity.

"Desalting" refers to the separation of a protein sample (including a protein conjugate compound with an agent) from the non-volatile salts, excipients and / or surfactants.

A "volatile salt" refers to a salt that enters the gaseous phase at the pressure of the surrounding environment (1 atmosphere). Volatile salts include, for example, ammonium formate, ammonium acetate, ammonium carbonate.

A "surfactant" includes, but is not limited to, non-volatile matrix compounds. For example, the surfactants include non-ionic or zwitterionic detergents, such as polysorbate 20 ("Tween 20") and polysorbate 80 ("Tween 80").

A "caotropic agent" refers to a chemical product that destabilizes hydrogen bonds, van der Waals forces and hydrophobic interactions between proteins and between subdomains of a protein and subsequently results in denaturation. Exemplary compounds include, but are not limited to, guanidine-HCl, urea, and other compounds known to the person skilled in the art.

An "excipient" includes compounds such as sugars and polyols, for example, sucrose, trehalose and sorbitol.

"Mass spectrometry" (MS) is an analytical technique that is used to measure the mass ratio with respect to the charge of charged particles. It can be used to deduce the primary structure of proteins.

"Kquida Chromatography" or "LC" refers to a method of separation of mixtures. The mixture dissolves in a fluid called the mobile fas that transports it through a structure called the stationary phase. In the case of size exclusion chromatography (SEC) the different constituents of the mixture travel at different speeds, causing them to separate. The separation is based on the differential partition between the stationary and mobile phases.

"Chromatography of exclusion by size" (SEC) refers to a method of chromatography in which the molecules are separated by their size and not by another separation parameter such as molecular weight or polarity. It is applied to large molecules such as proteins. In exclusion chromatography by size, the salts are used primarily as a mobile phase. In some cases, organic and caotropic agents may also be present in the mobile phase. The stationary phase is usually, but not limited to, one of the following, cross-linked polystyrene, derived from silica, acrylic, hydroxylated acrylic, acrylic, agarose, or a polyhydroxyethyl-aspartamide structure. The resin framework can be derived with any number of species and the choice of derivatizing agent is normally directed by a need to reduce undesirable molecular interactions between the analyte to be separated and the column that would be used in a separation method. based on a separation parameter different from the size of the compound.

As used herein, the phrase "volatile SEC buffer compatible with mass spectrometry" refers to the mobile phase of a SEC that is compatible for use in an MS.

As used herein, the term "antibody" refers in its broadest sense to an antibody, as used in the relevant field to which the present invention pertains, and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies ( for example, bispecific antibodies), and antibody fragments. The antibodies can be murine, human, humanized, chimeric, or derived from other species.

As used herein, the term "antibody" also refers to a full-length immunoglobulin molecule or an immunologically active part of a full-length immunoglobulin molecule, i.e., a molecule that contains a binding site. An antigen that binds to an antigen immunospecifically of a target of interest or a portion thereof, said targets include, but are not limited to, cancer cells, or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (eg, IgG, IgE, IgM, IgD, and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses of the molecule of immunoglobulin.

As used herein, the terms "antibody fragments" include, but are not limited to, a portion of a full-length antibody, generally the region of antigen binding or variable thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv; Fc, Fc means (1/2 Fc), diabodies; linear antibodies, fragments produced by an expression library of Fab, anti-idiotypic antibodies (anti-Id), CDR (complementarity determining region), ECD (extracellular domain), and fragments of attachment to the epltope of any of the foregoing which are an immunospecphically to cancer cell antigens, viral antigens, or microbial antigens; single chain antibody molecules; and multispecific antibodies formed by antibody fragments.

As used herein, an "intact antibody" refers to an antibody that includes the VH and VL domains, as! as the complete constant heavy and light chain domains and remain associated by at least one non-covalent interaction. An "intact antibody fragment" includes a pare of a full-length antibody that is associated by at least one non-covalent interaction. The term "interchain disulfide bond", in the context of an antibody, refers to a disulfide bond between two heavy chains, or a heavy and a light chain.

The term "intrachain disulfide bond", in the context of the antibody, refers to a disulfide bond formed from 2 cysteine residues in the same polypeptide chain.

The term "intercatenary thiol" refers to a thiol group, of an antibody heavy and light chain that can participate in the formation of an interchain disulfide bond.

The term "monoclonal antibody" or "mAb" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible mutations of natural origin which may be present in smaller quantities. The monoclonal antibodies are highly specific, being directed against a single antigenic site. Additionally, unlike with polyclonal antibody preparations that include different antibodies directed against different determinants (epltopos), each monoclonal antibody is directed against a single determinant in the antigen.

The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous with corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody , while the rest of the chain (s) is (are) identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibody, thus! as to fragments of said antibodies. The intact antibody can have one or more "effector functions" that refer to the activities that can be attributed to the Fc region (a native sequence of the Fc region or an amino acid sequence of a variant region of Fc) of an antibody. Examples of antibody effector functions include, but are not limited to, C1q binding; complement-dependent cytotoxicity; binding to the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and negative regulation of cell surface receptors (e.g., B cell receptor, BCR).

The antibody can be a fusion protein of an antibody, or a functionally active fragment thereof. For example, the antibody can be fused by a covalent bond (eg, a peptide bond), at the N-terminus or the C-terminus with respect to an amino acid sequence from another protein (or part thereof, such as at least one part of 10, 20 or 50 amino acids of the protein) that is not the antibody. The antibody or fragment thereof can be covalently linked to the other protein at the N-terminus of the constant domain.

The term "fusion protein" as used herein can also be referred to in the context of Ig-domain domain fissions in which the binding domain can be, for example, a ligand, an extracellular domain of a receptor , a peptide, a peptide not of natural origin or the like, provided that the binding domain does not include a variable domain of an antibody. Like the proteins and antibodies described herein, the Ig portion of the fusion protein must comprise at least one reducible disulfide bond. In one aspect The Ig domain will be the Fc region of an antibody. Examples of Ig-domain fusion proteins include etanercept which is an antibody protein of sNTFRII with the Fc region (U.S. Pat. No. 5,605,690), alefarcept which is a fusion protein of LFA-3. expressed in the antigen presenting cells with the Fc region (US Pat. No. 5,914,111), a fusion protein of the antigen-4 associated with the cytotoxic T lymphocyte (CTLA-4) with the Fc region (J Exp. Med. 181: 1869 (1995)), a fusion protein of interleukin 15 with the Fc region (J. Immunol. 160: 5742 (1998)), a fusion protein of factor VII with the Fc region (Proc. Natl. Acad. Sci. LISA 98: 12180 (2001)), a fusion protein of interleukin 10 with the Fc region (J. Immunol. 154: 5590 (1995)), a fusion protein of interleukin 2 with the region Fc (J. Immunol., 146: 915 (1991)), a fusion protein of CD40 with the Fc region (Surgery 132: 149 (2002)), a fusion protein of Flt-3 (fms-type tyrosine kinase) with the Fc region of the antibody (Acta. Haeraato 95:21 8 (1996)), a fusion protein of OX40 with, the Fc region of the antibody (J. Leu Biol. 72: 522 (2002)), and fusion proteins with other CD molecules (e.g., CD2, CD30 (TNFRSF8), CD95 (Fas), CD106 (VCAM-1), CD137), adhesion molecules (for example, ALCAM (activated cell leukocyte adhesion molecule), cadherins, ICAM (intercellular adhesion molecule) -1, ICAM-2, ICAM -3), cytokine receptors (e.g., interleukin-4R, interleukin-5R, interleukin-6R, interleukin-9R, interleukin-10R, interleukin-12R, interleukin-13R alpha 1, interleukin-13R alpha 2, interleukin-15R , interleukin-21Ralpha), chemokines, signal molecules that induce cell death (for example, B7-H1, DR6 (deadly receptor 6), PD-1 (programmed death-1), TRAILR1), co-stimulant molecules (for example, B7-1, B7-2, B7-H2, ICOS (inducible coestimulant)), growth factors (e.g., ErbB2, ErbB3, ErbB4, HGFR), differentiation-inducing factors (e.g., B7-H3), activating factors (e.g., NKG2D), signal transfer molecules (e.g., gpBO), BCMA, and TACI.

The abbreviation "MMAE" refers to monomethyl auristatin E.

The abbreviation "MMAF" refers to dovalin-valine-dolaisoleucine-dolaproline-phenylalanine, which is also referred to as monomethyl auristatin F.

The term "label" means a moiety that can be covalently linked to an antibody and which functions to: (i) provide a detectable signal; (ii) interacting with a second marker to modify the detectable signal provided by the first or second marker, for example FRET (energy transfer in fluorescence resonance); (iii) stabilizing the interactions or increasing the binding affinity with the antigen or the ligand; (iv) affect mobility, for example, electrophoretic mobility, or cell permeability, charge, hydrophobicity, shape or other physical parameters, or (v) provide a capture moiety, to modulate the affinity of the ligand, the antibody binding / antigen, or the formation of ionic complexes.

The antibodies can be conjugated to any marker moiety that can be covalently bound to the antibody by the thiol of the cysteine. For diagnostic applications, the antibody will normally be labeled with a detectable moiety.

The "drug" or "pharmacological moiety" can be any cytotoxic, cytostatic or immunomodulatory drug (e.g., immunosuppressant). In many cases, the drugs are conjugated to the antibody by a linker. For example, linkers are disclosed in U.S. Pat. UU No. 7,754,681; 7.375078; 7,829,531; 7,659,241; 7,851,437; 7,829,531; 7,659,241; 7,498,298; 7,994,135; 7,964,567 and 7,964,567; each of which is incorporated herein by reference in its entirety and for all purposes. In one aspect, the linker is valine-Citrulline ("Val-Cit" or "vc"). In another aspect, the linker is maleimidocaproyl ("mc").

II. Methods

Reference will now be made in detail to certain embodiments.

One skilled in the art will recognize that many methods and materials similar or equivalent to those described herein could be used in the practice of the present disclosure. The present disclosure is not limited to the methods and materials described.

In some embodiments, the present disclosure provides methods for the detection of a mass of a protein complex compound with an agent. In certain embodiments, the methods include the following steps: (a) providing a conjugate compound of a protein with a non-denatured agent and non-covalently associated in a volatile salt and free of a non-volatile salt; (b) introducing the conjugate compound of a protein with an agent in a mass spectrometer; and (c) directly establishing the mass of the conjugate compound of a protein with an agent by mass spectrometry.

In some embodiments, the present disclosure provides methods for the detection of a mass of a conjugate compound of a protein with a non-covalently associated agent. In certain embodiments, the methods include the following steps: (a) providing a conjugate compound of a protein with an agent in a volatile salt and free of a non-volatile salt; (b) introducing the conjugate compound of a protein with an agent in a mass spectrometer; and (c) directly establishing the mass of the conjugate compound of a protein with an agent by mass spectrometry.

In some of the methods described herein, the methods additionally include the application of a separation medium under non-denaturing conditions for the conjugate compound of a protein with an agent to effect the separation of the conjugate compound from a protein with a protein. agent of a matrix, whereby the conjugate compound of a protein with an agent is substantially undenatured. In other embodiments, the methods include the step of introducing a separation medium under non-denaturing conditions for the conjugate compound of a protein and an agent for effecting separation of the conjugate compound from a protein with an agent of a matrix, so that the conjugate compound of a protein with an agent is substantially non-denatured.

In some embodiments of the methods described herein, the method includes eluting the separation medium of a conjugate compound of a protein with an agent. In certain embodiments, the non-denaturing conditions include a volatile SEC buffer compatible with mass spectrometry. In some additional embodiments, the volatile salt includes ammonium acetate, ammonium acetate. In certain other embodiments, the volatile salt is ammonium acetate. In other embodiments, the volatile salt is ammonium carbonate. In certain other embodiments, the volatile salt is a mixture of the volatile salts discussed herein.

In some embodiments, the present disclosure provides methods, described herein, wherein the matrix includes a volatile salt, a surfactant, or a buffer.

In further embodiments of the methods set forth herein, the present disclosure provides methods that include the quantification of the relative distribution of conjugates compounds of a protein with an agent by ionized intensity broken down. In some of these embodiments, the methods include the quantification of the relative distribution of the protein conjugate compounds with an agent. In some of these embodiments, the methods include the breakdown of the intensity of an assay described herein in order to quantify the relative distribution of the conjugate compounds of a protein with an agent.

In further embodiments of the methods set forth herein, the conjugate compound of a protein with an agent includes a conjugate of an antibody to a drug.

In additional embodiments of the methods set forth herein, the matrix includes a formulation.

In certain embodiments, the matrix is a formulation.

In additional embodiments of the methods set forth herein, the separation medium includes size exclusion chromatography (SEC). In certain embodiments, the separation medium is size exclusion chromatography (SEC) A. In certain embodiments described herein, the methods include the step of separating conjugates of a protein with an agent, which are set forth herein, by exclusion chromatography by size.

In additional embodiments of the methods set forth herein, the agent includes a drug, a toxin, or a label. In some embodiments, the agent is a drug. In some other embodiments, the agent is a toxin. In some other embodiments, the agent is a marker. In certain embodiments, the agent is a combination of any of the agents set forth herein.

In further embodiments of the methods set forth herein, the SEC includes a column of silica, polystyrene-divinylbenzene or polyhydroxyethyl-aspartamide. In some embodiments, the SEC includes a column of silica. In some other embodiments, the SEC includes a polystyrene-divinylbenzene column. In some other embodiments, the SEC includes a polyhydroxyethyl-aspartamide column.

In further embodiments of the methods set forth herein, the non-denaturing conditions include a temperature of 50 ° C. In certain embodiments, the temperature of the non-denaturing conditions is not greater than 50 ° C. In some embodiments, the temperature of the non-denaturing conditions is an ambient temperature. Ambient temperature includes, but is not limited to, temperatures of 20-24 ° C.

In further embodiments of the methods set forth herein, mass spectrometry is carried out in an ESI-MS. In other embodiments, mass spectrometry is carried out in a mass spectrometer attached to another device such as a chromatograph or spray nozzle for the delivery of the sample to be analyzed in the MS.

In additional embodiments of the methods set forth herein, the concentration of the volatile salt is 50 to 400 mM. In some embodiments, the concentration of the volatile salt of 50 mM. In some embodiments, the concentration of salts is 400 mM. In some embodiments, the concentration of the volatile salt is 50, 55,

60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,

185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, or 400 mM. In some embodiments, the concentration of the volatile salt is about 50 to about 400 mM. In this context, approximately refers to a value that is between 10% of the value modified by the word approximately. For example, approximately 50 mM, includes 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55 mM.

For example, approximately 400 mM includes 360, 370, 380, 390, 400, 410, 420, 430, and 440 mM. In some embodiments, the concentration of the volatile salt is about 50 to about 300 mM. In some embodiments, the volatile salt concentration is about 50 mM to about 200 mM. In some embodiments, the concentration of the volatile salt is about 50 to about 100 mM. In some embodiments, the concentration of the volatile salt is about 100 to about 400 mM. In some embodiment, the concentration of the volatile salt is about 200 to about 400 mM. In some embodiments the volatile salt concentration is about 300 to about 400 mM. In some embodiments, the volatile salt concentration is about 50 to about 100 mM. In some embodiments, the concentration of the volatile salt is from about 50 to about 200 mM. In some embodiments, the concentration of the volatile salt is about 50 to about 300 mM. In some embodiments, the concentration of the volatile salt is from about 50 to about 350 mM.

In additional embodiments of the methods set forth herein, the conjugate compound of a protein with an agent is immediately introduced into the mass spectrometer.

In some embodiments of the methods set forth herein, the eluted compound of a protein conjugate with an agent is continuously introduced into the mass spectrometer. In certain embodiments, the separation medium is a column of HIC that is run under non-denaturing conditions.

In other embodiments, the SEC is a polyhydroxyethyl-A column.

In some embodiments of the methods set forth herein, the size of the SEC column is 0.1 to 7.8 mm internal diameter and 100 to 300 mm long.

In further embodiments of the methods set forth herein, the methods include the step of treating the conjugate compound with a protein with an agent with a deglycosylating reagent.

In certain embodiments, the deglycosylating reagent is PNGase F. In other embodiments, the deglycosylating agent includes an enzymatic treatment with an exoglycosidase, a treatment with an endoglycosidase, or an enzymatic treatment that makes a split between the 1st and 2nd residues of N- acetylglucosamine in the N-glycans.

In some of the embodiments described herein, treatment with exoglycosidase includes sialidase or beta-galactosidase.

In further embodiments of the methods set forth herein, the enzymatic treatment that cleaves between the 1st and 2nd N-acetylglucosamine residue of the N-glycans includes Endo-F1, F2 or F3.

In some additional embodiments of the methods set forth herein, the pH of the volatile salt is from 5.5 to 7.0. In other embodiments, the methods described herein include the step wherein the pH of the volatile salt is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6, 1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some embodiments of the methods set forth herein, the pH of the volatile salt is 6.0. In some embodiments of the methods set forth herein, the pH of the volatile salt is 6.5. In some embodiments of the methods set forth herein, the pH of the volatile salt is 7.0.

In additional embodiments of the methods set forth herein, the methods include the quantification of the non-denatured compound of a protein conjugate and an agent by mass spectrometry.

In some embodiments of the methods set forth herein, the non-denatured conjugate compound of a protein and an agent includes a heavy chain or antibody light chain fragment. In some of these embodiments, the heavy chain or antibody light chain fragment further includes one or more drugs.

In additional embodiments of the methods set forth herein, the antibody to the antibody and drug conjugate is an antibody fragment.

In further embodiments of the methods set forth herein, the antibody fragment is selected from a Fab, Fab ', F (ab') 2, Fv, diabody, linear antibody, or single chain antibody molecule fragment. . In some of the embodiments described herein, the antibody is selected from the group consisting of an anti-CD30, anti-CD40, Anti-CD19, anti-CD33 or anti-CD70 antibody. In other embodiments, the antibody of the conjugate of an antibody to a drug is a humanized antibody. In some embodiments the antibody of the conjugate of an antibody to a drug includes a humanized antibody.

In additional embodiments of the methods set forth herein, the drug is a maytansinoid.

In some other embodiments, the drug is an auristatin. In certain embodiments, the drug is MMAE. In some embodiments, the drug is MMAF.

In additional embodiments of the methods set forth herein, the mass of the conjugate compound of a protein and an agent is measured at 7.5 Da. In other embodiments, the mass of the conjugate compound of a protein and an agent is measured at 25 Da. In certain embodiments, the mass of the conjugate compound of a protein and an agent is measured at 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7. , 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9 , 0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2 , 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11 , 5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7 , 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15.0, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16.0, 16.1, 16.2, 16.3, 16.4, 16.5, 16.6, 16, 7, 16.8, 16.9, 17.0, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 0, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19.0, 19.1, 19.2, 19, 3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20.0, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21.0, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22.0, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23.0, 23.1, 2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24.0, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25.0, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25, 7, 25.8, or 25.9 Da.

In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 8.0 Da. In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 9.0 Da. In additional embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 10.0 Da. In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 11.0 Da. In additional embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 12.0 Da. In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 13.0 Da. In additional embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 14.0 Da. In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 15.0 Da. In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 16.0 Da. In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 17.0 Da. In additional embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 18.0 Da. In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 19.0 Da. In additional embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 20.0 Da. In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 21.0 Da. In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 22.0 Da. In additional embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 23.0 Da. In further embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 24.0 Da. In additional embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 25.0 Da.

In additional embodiments of the methods set forth herein, the mass of the conjugate compound of a protein with an agent is measured at 100 ppm of the theoretical value. In other embodiments, the mass of the conjugate compound of a protein with an agent is measured at 80, 90, 100, 110, 120, or 130 ppm of the theoretical value.

The present disclosure discloses methods for testing intact native mass of conjugates of a protein with an agent. The present disclosure also discloses methods for determining the intact native mass of conjugates of a protein with an agent.

The present disclosure discloses a method for detecting the mass of a conjugate compound of a protein with a non-covalently associated agent by providing a conjugate compound of a protein with a non-denatured agent in a volatile salt and free of non-volatile salts; introducing the conjugate compound of a protein with an agent in a mass spectrometer; and directly establishing the mass of the conjugate compound of a protein with an agent by mass spectrometry.

The present disclosure provides methods in which the conjugate of a protein with an agent is introduced into a mass spectrometer. In more of these embodiments, the mass of the conjugate compound of a protein with an intact agent is measured directly. In one of these embodiments, the conjugate compound of a protein with an agent is non-denatured and in ammonia form. In some of these embodiments, the conjugate compound of a protein with an agent is non-denatured and in ammonium acetate. In other of these embodiments the conjugate compound of a protein with an agent is non-denatured and in ammonium carbonate. In some of these embodiments, the conjugate compound of a protein with an agent is a conjugate compound of an antibody with an agent that is non-denatured and in ammonium carbonate. In some of these embodiments, the conjugate compound of a protein with an agent is a conjugate compound of an antibody with an agent that is not denatured and in ammonium carbonate. In some of these embodiments, the conjugate compound of a protein with an agent is a conjugate compound of an antibody with an agent that is non-denatured and in ammonia form. In some of these embodiments, the conjugate compound of a protein with an agent is an antibody conjugate compound with a agent that is non-denatured and in ammonium acetate.

The present disclosure provides methods in which a conjugate of a protein with an agent is introduced into a mass spectrometer. In more of these embodiments, the mass of the conjugate compound of a protein with an intact agent is measured directly. In some other embodiments, the conjugate compound of a protein and an agent is a conjugate compound of an antibody fragment with an agent, which is not denatured and in ammonium carbonate. In some of these embodiments, the conjugate compound of a protein with an agent is a conjugate compound of an antibody fragment with an agent that is not denatured and in ammonia form. In some of these embodiments, the conjugate compound of a protein with an agent is a conjugate compound of an antibody fragment with an agent that is not denatured and in ammonium acetate.

The present disclosure provides methods in which the conjugate of a protein with an agent is introduced into a mass spectrometer. In more of these embodiments, the mass of the conjugate compound of a protein with an intact agent is measured directly. In some of these embodiments, the conjugate compound of a protein with an agent is non-denatured. In some of these embodiments, the conjugate of a protein with an agent is in ammonia form and the agent is a drug. In some of these embodiments, the conjugate of a protein and an agent is in ammonia form and the agent is a drug. In some embodiments, the conjugate of a protein with an agent is in ammonium carbonate and the agent is a drug. In any of these embodiments, the drug includes MMAE or MMAF.

In related embodiments, the methods described herein provide that the conjugate compound of a protein with an agent is a conjugate compound of an antibody with an agent that is non-denatured. In some of these embodiments, the conjugate of a protein with an agent is in ammonium carbonate and the agent is a drug. In some of these embodiments, the conjugate of a protein with an agent is in ammonia form and the agent is a drug. In some of these embodiments, the conjugate of a protein with an agent is in ammonium acetate and the agent is a drug. In some embodiments, the drug includes, but is not limited to, MMAE or MMAF. In any of these embodiments, the drug includes MMAE. In some of these embodiments, the drug is MMAF.

The present disclosure provides methods in which the conjugate of a protein with an agent is introduced into a mass spectrometer. In more of these embodiments, the mass of the conjugate compound of a protein with an intact agent is measured directly. In some of these embodiments, the conjugate compound of a protein with an agent is non-denatured. In some embodiments, the conjugate of a protein with an agent is in ammonia form and the agent is a toxin. In some other embodiments, the conjugate of a protein with an agent is in ammonium carbonate and the agent is a toxin. In some embodiments, the conjugate of a protein with an agent is in ammonium acetate and the agent is a toxin. In some of these embodiments, the agent is a toxin. In any of these embodiments, the agent is a drug.

The present disclosure provides methods in which the protein conjugate with an agent is introduced into a mass spectrometer. In more of these embodiments, the mass of the conjugate compound of a protein with an intact agent is measured directly. In some of these embodiments, the conjugate compound of a protein with an agent is a conjugate compound of an antibody with an agent that is not denatured. In some embodiments, the salt concentration is between 50 and 400 mM. In some of these embodiments, the agent is a drug. In some of these embodiments, the conjugate of a protein with an agent is in ammonium carbonate, ammonium acetate, or ammonium formate.

In any of the embodiments set forth herein, the methods may include the step in which the conjugate compound is introduced into a mass spectrometer, and the mass of the antibody conjugate with an intact agent is measured directly.

The present disclosure provides methods in which the conjugate of a protein with an agent is introduced into a mass spectrometer. In more of these embodiments, the mass of the conjugate compound of a protein and an intact agent is measured directly. In some embodiments, the conjugate compound of a protein with an agent is a conjugate compound of a protein with an agent that is not denatured. In related embodiments, the protein conjugate with an agent is in ammonium acetate, ammonium carbonate, or in ammonium form. In some embodiments, the concentration of salts is between 50 and 400 mM. In some embodiments, the salt concentration is 200 mM. In other related embodiments, the agent is a drug. In other embodiments, the pH of the buffer is between 5.0 and 7.0. In any of these embodiments, the agent can be a drug.

In another aspect of the methods described herein, the conjugate compound of a protein with an agent is a conjugate compound of an antibody with an agent that is not denatured and in ammonia; the salt concentration is 200 mM; the agent is a drug; and the pH of the buffer is between 5.0 and 7.0. In some of these embodiments, the protein conjugate with an agent is in ammonium carbonate, ammonium acetate, or in ammonium form.

In another aspect, the conjugate compound of a protein with an agent that is not denatured and in ammonium acetate. In some embodiments, the salt concentration is 200 mM; the pH of the buffer is between 5.0 and 7.0; and the agent is a drug.

In another aspect of the methods of the present document, the conjugate compound of a protein with an agent is a conjugate compound of an antibody with an agent that is not denatured, the concentration of salts is 200 mM; the agent is a drug; and the pH of the buffer is 6.5. In some of these embodiments, the conjugate of a protein with an agent is in ammonium carbonate, ammonium acetate, or in ammonium form. In some of these embodiments, the agent includes a drug, wherein the drug is a drug described herein.

In one aspect of the present disclosure the conjugate compound of a protein with an agent is undenatured, a means of separation under non-denaturing conditions is applied to the conjugate compound of a protein with an agent in a matrix to effect the separation of the composed of a conjugate of a protein with a matrix agent. In some embodiments, the conjugate compound of a protein with an agent is substantially non-denatured, the conjugate is introduced into a mass spectrometer, and the mass of conjugate compound of a protein with an intact agent is measured directly. In some of these embodiments, the conjugate of a protein with an agent is in ammonium carbonate, ammonium acetate, or ammonium formate.

The present disclosure provides a method in which a conjugate of a protein with an agent is introduced into a mass spectrometer. In more of these embodiments, the mass of the conjugate compound of a protein with an intact agent is measured directly. In another aspect of the methods set forth herein, the c-conjugate compound of a protein with an agent is non-denatured, and a means of separation under non-denaturing conditions is applied to the conjugate compound of a protein with a non-denaturing agent. a matrix for effecting the separation of the conjugate compound from a protein with an agent of the matrix, whereby the conjugate compound of a protein with an agent is substantially undenatured. In some of these embodiments, the conjugate compound of a protein with an agent is in ammonium carbonate. In some of these embodiments, the conjugate compound of a protein with an agent is in ammonium acetate. In related embodiments, a means of separation under non-denaturing conditions is applied to the protein conjugate in a matrix to effect separation of the conjugate compound from a protein with a matrix agent. In related embodiments, the separation means is SEC.

In any of the above methods, the methods may include that the conjugate compound of a protein with an agent is non-denatured, a means of separation under non-denaturing conditions is applied to the conjugate compound of a protein with an agent in a matrix to effect the separation of the conjugate compound from a protein with a matrix agent, whereby the conjugate compound of a protein with an agent is substantially undenatured, and the separation medium is HCl under non-denaturing conditions. In related embodiments, the conjugate of a protein with an agent is in ammonium carbonate. In related embodiments, the conjugate of a protein with an agent is in ammonium acetate. In related embodiments, the protein conjugate with an agent is in ammonic format.

In any of the above methods, the methods may include that the conjugate compound of a protein with an agent is non-denatured, a means of separation under non-denaturing conditions is applied to the conjugate compound of a protein with an agent in a matrix to effect the separation of the conjugate compound from a protein with a matrix agent, whereby the conjugate compound of a protein with an agent is substantially non-denatured, and the separation medium is HCl under non-denaturing conditions. In related embodiments, the conjugate of a protein with an agent is in ammonium carbonate, ammonium formate, or ammonium acetate. In any of the methods described herein, the separation medium may include SEQ.

In another aspect of the foregoing methods, the conjugate compound of a protein with an agent is a conjugate compound of an antibody with an agent, is non-denatured, a means of separation is applied under non-denaturing conditions to the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound from an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the separation medium is SEC. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In one aspect of the methods in which the conjugate of a protein with an agent is introduced into a mass spectrometer, and the mass of the conjugate compound of an antibody with an intact agent is measured directly, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, is non-denatured, a separation medium is applied under non-denaturing conditions for the conjugate compound of an antibody and an agent in a matrix to effect the separation of the compound from complex of an antibody with a matrix agent, so the compound of The conjugate of an antibody with an agent is substantially non-denatured, and the separation medium is HIC under non-denaturing conditions. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In one aspect of the methods in which the protein conjugate is introduced into a mass spectrometer, and the mass of the conjugate compound of an antibody with an intact agent is measured directly, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, is non-denatured, a means of separation under non-denaturing conditions is applied to the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound from an antibody with an agent of the matrix, whereby the conjugate compound of an antibody with an agent is substantially non-denatured, and the separation medium is HIC under non-denaturing conditions. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate. In related embodiments, the separation medium is SEC and the mass spectrometer is an ESI-MS.

In one aspect of the above methods, the conjugate compound of a protein with an agent is a conjugate compound of an antibody with an agent, is non-denatured, a means of separation is applied under non-denaturing conditions to the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound from an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the separation medium is SEC, the conjugate is introduced into a mass spectrometer, the mass spectrometer is an ESI-MS, and the mass of the conjugate compound of an antibody with an intact agent is measured directly. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In another aspect of the above methods, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, is non-denatured, a means of separation is applied under non-denaturing conditions to the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound from an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the separation medium is SEC. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In one aspect of the above methods, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, the conjugate is non-denatured, a means of separation is applied under non-denaturing conditions to the compound of conjugate of an antibody and an agent in a matrix to effect the separation of the complex compound of an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the Separation is HIC under non-denaturing conditions, the conjugate is introduced in a mass spectrometer and the mass spectrometer is an ESI-MS. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In any of the methods described herein, the methods may include the step in which the mass of the conjugate compound of an antibody with an intact agent is measured directly. In any of the methods described herein, the protein conjugate with an agent can be present in ammonium carbonate, in ammonium form, or in ammonium acetate.

In another aspect of the above methods, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, is non-denatured, a means of separation is applied under non-denaturing conditions to the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound from an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the separation medium is HIC under non-denaturing conditions. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate. In related embodiments, the mass spectrometer is an ESI-MS. In some other related embodiments, the conjugate of a protein with an agent is continuously introduced into a mass spectrometer.

In any of the methods described herein in which the conjugate of a protein with an agent is introduced into a mass spectrometer, the conjugate can be introduced continuously. In any of the methods described herein in which the conjugate of a protein with an agent is introduced into a mass spectrometer, the conjugate can be introduced continuously.

In another aspect of the above methods, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, is non-denatured, a means of separation is applied under non-denaturing conditions to the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound of an antibody with an agent from the matrix, whereby the The conjugate of an antibody with an agent is substantially undenatured, and the separation medium is SEC, and the mass spectrometer is an ESI-MS. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In one aspect of the above methods, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, it is non-denatured, a separation medium is applied under non-denaturing conditions to the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound from an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the separation medium is HIC under non-denaturing conditions. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In any of the methods described herein, the mass spectrometer may include an ESI-MS.

In another aspect of the above methods, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, is non-denatured, a means of separation is applied under non-denaturing conditions to the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound from an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the separation medium is SEC, and the mass spectrometer is ESI-MS. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

The present disclosure provides methods in which the separation medium is SEC, the protein conjugate is introduced into a mass spectrometer, the mass spectrometer is an ESI-MS, and the mass of the compound of an antibody conjugate with an agent intact is measured directly and the mass of the compound of an antibody conjugate with an agent is quantified. In another aspect, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, which is not denatured, a separation medium is applied under non-denaturing conditions to the conjugate compound of an antibody and a agent in a matrix to effect the separation of the complex compound of an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially non-denatured In related embodiments, the protein conjugate with an agent it is in ammonium carbonate, in ammonium form, or in ammonium acetate. In other related embodiments, the separation medium is HIC under non-denaturing conditions.

In one aspect, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, the conjugate of a protein with an agent is non-denatured, a means of separation is applied under non-denaturing conditions for the agent. conjugate of an antibody and an agent in a matrix to effect the separation of the complex compound of an antibody with an agent of the matrix, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the Sequestration medium is SEC, SEC column is silicon, polystyrene-divinylbenzene or polyhydroxyethylaspartamide, and the compound of an antibody conjugate with an agent is introduced in a mass spectrometer, and the mass of the compound of an antibody conjugate with an intact agent is measured directly. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In one aspect of the above methods, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, the conjugate of a protein with an agent is non-denatured, a means of separation is applied under conditions non-denaturing for the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound of an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially not denatured, and the means of separation is SEC. In related embodiments, the SEC column is of silica. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate. In other related embodiments, the SEC includes polystyrene-divinylbenzene.

In any of the methods of the present document in which the antibody conjugate with an agent is introduced into a mass spectrometer, and the mass of the conjugate compound of an antibody with an intact agent is measured directly, the methods may additionally include the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, is non-denatured, a means of separation under non-denaturing conditions is applied to the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound of an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the separation medium is SEC, and the SEC column is of polyhydroxyethyl aspartamide. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In any of the methods of the present document in which the antibody conjugate with an agent is introduced into a mass spectrometer, and the mass of the conjugate compound of an antibody with an intact agent is measured directly, the methods may additionally include following aspects. In another aspect, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, is non-denatured, a separation medium is applied under non-denaturing conditions to the conjugate compound of an antibody and a agent in a matrix to effect the separation of the complex compound from an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the separation medium is SEC, and the SEC column is polyhydroxyethyl aspartamide. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In related embodiments, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, the conjugate of a protein with an optionally deglycosylated agent, the conjugate of a protein with an agent is not denatured, a means of separation under non-denaturing conditions is applied to the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound of an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially non-denatured, the separation medium is s Ec, and the SEC column is silica, polystyrene-divinylbenzene or polyhydroxyethylaspartamide. In related embodiments, the protein conjugate with an agent is in ammonium carbonate, in ammonium form, or in ammonium acetate.

In another aspect of the methods in which the antibody conjugate with an agent is introduced into a mass spectrometer, and the mass of the conjugate compound of an antibody with an intact agent is measured directly, the methods may include the following aspects. In another aspect, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, the conjugate of a protein with an agent in a compound of an antibody conjugate with an agent is deglycosylated, the conjugate of a protein with an agent is non-denatured, a means of separation is applied under non-denaturing conditions for the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound of an antibody with an agent of the matrix, whereby the conjugate compound of an antibody with an agent is substantially undenatured, the separation medium is SEC, and the SEC column is of silica, polystyrene-divinylbenzene or polyhydroxyethyl-aspartamide. In related embodiments the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, it is non-denatured. In related embodiments, the protein conjugate with an agent is in ammonium carbonate and the ammonium carbonate concentration is between 50 and 400 mM. In other embodiments, the protein conjugate with an agent is in ammonium format and the ammonium formate concentration is between 50 and 400 mM.

In any of the methods described herein, the methods can provide that the conjugate of a protein with an agent is in ammonium carbonate and the concentration of ammonium carbonate is between 50 and 400 mM. In any of the methods described herein, the methods can provide that the conjugate of a protein with an agent is in ammonium acetate and the concentration of ammonium acetate is between 50 and 400 mM. In any of the methods described herein, the methods can provide that the conjugate of a protein with an agent is in ammonium format and the concentration of the ammonium format is between 50 and 400 mM.

In another aspect of the methods in which the protein conjugate is introduced into a mass spectrometer, and the mass of the conjugate compound of an antibody with an intact agent is measured directly, the methods may include the following aspects. In one aspect the separation medium has non-denaturing conditions for the conjugate compound of an antibody and an agent in a matrix and is applied to effect the separation of the complex compound of an antibody with an agent from the matrix, whereby the When the conjugate of an antibody with an agent is substantially undenatured, the separation medium is SEC. In related embodiments the SEC column is of silica, polystyrene-divinylbenzene or polyhydroxyethyl-aspartamide. In any of the methods described herein, the methods can provide that the conjugate of a protein with an agent is in ammonium carbonate and the concentration of ammonium carbonate is between 50 and 400 mM. In any of the methods described herein, the methods can provide that the conjugate of a protein with an agent is in ammonium acetate and the concentration of ammonium acetate is between 50 and 400 mM. In any of the methods described herein, the methods can provide that the conjugate of a protein with an agent is in an ammonic format and the ammonium formate concentration is between 50 and 400 mM.

In related embodiments, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, the conjugate of a protein with an agent is non-denatured, a means of separation is applied under non-denaturing conditions for the agent. conjugate of an antibody and an agent in a matrix to effect the separation of the complex compound of an antibody with an agent of the matrix, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the Seperation medium is SEC, and SEC column is silica, polystyrene-divinylbenzene or polyhydroxyethyl-aspartamide. In related embodiments, the protein conjugate with an agent is in ammonic form at a concentration of between 50 and 400 mM and the pH is 6.5, the protein conjugate with an agent is in ammonium carbonate at a concentration of between 50 and 400 mM. and 400 mM and the pH of 6.5, or the protein conjugate with an agent is in ammonium acetate at a concentration of between 50 and 400 mM and the pH of 6.5. In other related embodiments, the protein conjugate with an agent is in ammonia form at a concentration of 250 mM and the pH of 6.5, the protein conjugate with an agent is in ammonium carbonate at a concentration of 250 mM and the pH of 6.5, or the protein conjugate with an agent is in ammonium acetate at a concentration of 250 mM and the pH of 6.5.

In any of the methods described herein, the methods may optionally include that the protein conjugate with an agent is in ammonia form at a concentration of 50 mM and the pH of 6.5, the protein conjugate with an agent is present. in ammonium carbonate at a concentration of 50 mM and the pH of 6.5, or the protein conjugate with an agent is in ammonium acetate at a concentration of 50 mM and the pH of 6.5.

In any of the methods described herein, the methods may optionally include that the protein conjugate with an agent is in ammonic form at a concentration of 150 mM and the pH of 6.5, the protein conjugate with an agent is present. in ammonium carbonate at a concentration of 150 mM and the pH of 6.5, or the protein conjugate with an agent is in ammonium acetate at a concentration of 150 mM and the pH of 6.5.

In any of the methods described herein, the methods may optionally include that the protein conjugate with an agent is in ammonia form at a concentration of 250 mM and the pH of 6.5, the protein conjugate with an agent is present. in ammonium carbonate at a concentration of 250 mM and the pH of 6.5, or the protein conjugate with an agent is in ammonium acetate at a concentration of 250 mM and the pH of 6.5.

In any of the methods described herein, the methods may optionally include that the protein conjugate with an agent is in ammonia form at a concentration of 400 mM and the pH of 6.5, the protein conjugate with an agent is present. in ammonium carbonate at a concentration of 400 mM and the pH of 6.5, or the protein conjugate with an agent is in ammonium acetate at a concentration of 400 mM and the pH of 6.5.

In another aspect of the methods of the present document in which the compound of an antibody conjugate with an agent is introduced into a mass spectrometer, the mass of the compound of an antibody conjugate with an intact agent is measured directly, and the spectrometer of masses is an ESI-MS, the methods may include the following aspects. In one aspect, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent and is not denatured. In related embodiments, the protein conjugate with an agent is in ammonia form at a concentration of 250 mM and the pH of 6.5, the protein conjugate with an agent is in ammonium carbonate at a concentration of 250 mM and the pH of 6.5, or the protein conjugate with an agent is in ammonium acetate at a concentration of 250 mM and the pH of 6.5.

In another aspect of the methods of the present document in which the compound of an antibody conjugate with an agent is introduced into a mass spectrometer, the mass of the compound of an antibody conjugate with an intact agent is measured directly, and the spectrometer of masses is an ESI-MS, the methods may include the following aspects. In one aspect, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, the conjugate of a protein with an agent is not denatured, a means of separation is applied under non-denaturing conditions for the agent. conjugate of an antibody and an agent in a matrix to effect the separation of the complex compound of an antibody with an agent of the matrix, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the separation medium is SEC, and the SEC column is polyhydroxyethyl aspartamide. In related embodiments the protein conjugate with an agent is in ammonia form at a concentration of 250 mM and the pH of 6.5, the protein conjugate with an agent is in ammonium carbonate at a concentration of 250 mM and the pH of 6. , 5, or the protein conjugate with an agent is in ammonium acetate at a concentration of 250 mM and the pH of 6.5.

In another aspect of the methods of the present document in which the compound of an antibody conjugate with an agent is introduced into a mass spectrometer, the mass of the compound of an antibody conjugate with an intact agent is measured directly, and the spectrometer of masses is an ESI-MS, the methods may include the following aspects. In one aspect, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent and is not denatured, a separation medium is applied under non-denaturing conditions to the conjugate compound of an antibody and a agent in a matrix to effect the separation of the complex compound from an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially undenatured, and the separation medium is SEC, and the SEC column is polyhydroxyethyl aspartamide. In related embodiments the protein conjugate with an agent is in ammonia form at a concentration of 250 mM and the pH of 6.5, the protein conjugate with an agent is in ammonium carbonate at a concentration of 250 mM and the pH of 6. , 5, or the protein conjugate with an agent is in ammonium acetate at a concentration of 250 mM and the pH of 6.5.

In some methods of the present document in which the compound of an antibody conjugate with an agent is substantially non-denatured, the separation medium is SEC, and the SEC column is polyhydroxyethylaspartamide. In related embodiments the protein conjugate with an agent is in ammonia form at a concentration of 250 mM and the pH of 6.5, the protein conjugate with an agent is in ammonium carbonate at a concentration of 250 mM and the pH of 6. , 5, or the protein conjugate with an agent is in ammonium acetate at a concentration of 250 mM and the pH of 6.5.

In another aspect of the methods of the present document in which the compound of an antibody conjugate with an agent is introduced into a mass spectrometer, the mass of the compound of an antibody conjugate with an intact agent is measured directly, and the spectrometer of masses is an ESI-MS, the methods may include the following aspects. In one aspect of a method described herein, the conjugate compound of a protein with an agent is a compound of an antibody conjugate with an agent, the agent is a drug, a means of separation is applied under non-denaturing conditions for the conjugate compound of an antibody and an agent in a matrix to effect the separation of the complex compound of an antibody with a matrix agent, whereby the conjugate compound of an antibody with an agent is substantially non-denatured, the separation medium is SEC, and the SEC column is polyhydroxyethylaspartamide. In related embodiments the protein conjugate with an agent is in ammonia form at a concentration of 250 mM and the pH of 6.5, the protein conjugate with an agent is in ammonium carbonate at a concentration of 250 mM and the pH of 6. , 5, or the protein conjugate with an agent is in ammonium acetate at a concentration of 250 mM and the pH of 6.5.

In some embodiments, the methods described herein include the rapid determination of the intact mass of the conjugate compounds of a protein with a non-covalently associated agent. In one embodiment a compound of an antibody conjugate with a heavy chain (HC) and light chain (LC) agent are present as a complex as a result of the reduction of the antibody and the subsequent conjugation of a drug in the interchain cysteine residues. . The methods described herein may include the step of analyzing the ADC using native desalination conditions, in which the bivalent and intact structure of the ADC is maintained. Additional embodiments include the stage at which the mass of the desalted ADC is subsequently determined using desolvation and ionization conditions. In some of these embodiments, the desolvation and ionization conditions are conventional desolvation and ionization conditions.

The methods described herein may include the analysis of ADCs with bound cysteines, presenting a method of mass measurement and desalination based on semi-native exclusion chromatography (SEC). The method can also include the measurement of the intact mass of the ad with intercatenary cysteines linked, and the quantification of the relative distribution of species with drugs bound by disaggregated ionic intensity. The method described herein can be adapted for the determination of high performance ADC masses.

The method described herein may include the optional deglycosylation step of the ADC. The deglycosylation step may include the use of PNGase F or other compounds known to the person skilled in the art. The methods described herein may include the step of eliminating glycan heterogeneity. In certain embodiments, the methods provide an enhanced, increased, or increased signal intensity of each drug species loaded. In additional embodiments, the methods include the quantification step. The present disclosure provides methods in which deglycosylation allows a better mass accuracy due to an increased MS signal intensity.

In addition to optional deglycosylation, the method of the present document may include a step of a separation medium. The step of the separation medium includes different chromatographic techniques, for example SEC or HIC. In this stage the ADC is exchanged from a buffer system to a volatile salt that supports the positive charge of basic amino acids for the measurement of the mass. In some of these embodiments, the non-volatile sels, surfactants and buffers of the ADC are removed. In other embodiments, the chromatographic step is carried out using the SEC under non-denaturing conditions, while allowing an exchange of ADC buffer in a volatile salt.

The method set forth herein may include a mass spectrometric stage. In certain aspects, the MS may be immediately following the separation medium stage. In other aspects, there may be stages of intervention between the separation medium stage and the MS stage. In still other aspects, the MS stage may be continuous following the medium separation stage. In the MS stage, the mass of the ADC is measured. Additionally, the methods set forth herein can provide that the quantitative drug loading is determined by an ion abundance MS. In one aspect, the MS is carried out in a TOF-MS, a Q-TQF, FTICR, Orbitrap, or high-resolution ion trap MS. In this stage, the mass is measured with a high-resolution mass spectrometer and the data of multiple load (m / z) is broken down to zero charge mass spectrum. Species loaded with individual drugs are quantified based on the ionic intensity of the disaggregated spectrum that is a product of the raw raw data intensity. The raw ESI-MS data of the proteins is normally displayed as a series of charged ions in which a single molecular species can have several ions associated with it and each of the related ions will differ in the number of positive charges. The breakdown of the raw data refers to the fact that the determination procedure of the number of charges of each ion is carried out and all the related ions are converted into a zero-charge mass spectrum that represents the molecular weight of the species that are being analyzed Each ion in raw data that relates to a particular species in the disaggregated mass spectrum has a measure of intensity or abundance associated with it, and the abundance of all ions associated with a particular species thus constitutes an approximation of the abundance of this species in the sample that is analyzed. When species are quantified, the abundance of a particular species in a sample is compared to the sum of the abundance of all related species in the sample giving as! a measure of the relative molar abundance of that particular species. The quantification in this way assumes in certain cases, that all species have approximately equivalent ionization efficiency.

III. Pharmacokinetics

Control of circulating levels of a therapeutic agent is necessary for pharmacokinetic (PK) determinations in a mammal, including half-life, clearance, area under the curve (AUC), and volume of distribution, to establish safety limits / Toxicity and appropriate dosage regimen (Welling, P. (1997) Pharmacokinetics Processes, Mathematics, and Applications, 2nd Ed., American Chemical Society, Washington, DC). Bioavailability is the extent to which the compound administered reaches the general circulation from the dosage form administered, usually expressed as a percentage of the dose administered. The half-life of a compound is the time required for the elimination of 505 of the peak concentration in the plasma of the compound by excretion or biotransformation (metabolism). The therapeutic index expresses the selectivity of the compound between the desired therapeutic activity and unwanted toxic side effects . Pharmacokinetic measurements clarify the absorption, distribution, metabolism, and excretion (ADME) of the conjugates of an antibody with a drug (ADC).

IV. Drug loading

The average number of drugs per antibody in the ADC preparations from the conjugation reactions can be characterized by the methods of the present disclosure. The methods can determine the amount of drug bound by antibody (loading) of an ADC and the distribution of drug moieties or fragments such as a heavy chain and a light chain.

V. Administration of antibody conjugates with a drug

The ADCs can be contacted with, or administered in, biological sources by any means appropriate to the condition to be treated. The ADC will normally be administered to a mammal by parenteral route, for example, in infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

SAW. Pharmaceuticals suitable for use in the methods described in this document

Exemplary drugs for conjugation with an antibody include drugs or cytotoxic agents particularly those used in cancer therapy. Such drugs include, in general, agents that damage DNA, anti-metabolites, natural products and their analogues. Exemplary classes of cytotoxic agents include enzyme inhibitors, such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNA cutters, topoisomerase inhibitors, anthracycline drug family, vinca drugs. , mitomycins, bleomycins, cytotoxic nucleosides, family of pteridine drugs, dinens, podophyllotoxins, dolastins, maytansinoids, inducers of differentiation, and taxol. Exemplary pharmacological moieties include, but are not limited to: auristatins (such as MMAF and MMAE), methotrexate, metopherin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosin, leurosidelna, actinomycin, daunorubicin, doxorubicin , mitomycin C, mitomycin A, maytansinoids, carmomycin, aminopterin, talisomycin, podophyllotoxin, and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere, retinoic acid, butyric acid, camptothecin, calicheamicin, Spiramycin, enedines, and their derivatives and analogues.

The pharmacological moiety of an ADC may also include dolastatin and its peptide analogs and derivatives, auristatins (U.S. Pat. No. 5,635,483; 5,780,588). It has been shown that dolastatins and auristatins interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) ^{Antimicrob Agents and Chemother 45 (12): 3580-3584) and have anticancer activities (US Pat} u nts Chemother ^42:... 2961-2965). In the U g . ^No. 5,663,149) and antifungals (Pettit et al (1998) Antimicrob In some embodiments, the drug is an auristatin, an anti-tubulin agent.The pharmacological moiety of dolastatin or auristatin can be bound to the antibody via the N-terminus. (amino) or the C-terminus (carboxyl) of the peptide drug moiety (WO 02/088172) The auristatin E variants are disclosed in U.S. Patent No. 5,767,237 and U.S. Pat. US No. 6,124,431.

The immunoconjugates of MMAE and MMAF, its synthesis and structure are disclosed in U.S. Pat. UU No. 7,851,437; 7,829,531; 7,659,241; 7,498,298; 7,994,135; 7,964,567; each of which is incorporated in the present document by reference in its entirety and for all purposes. Auristatins include but are not limited to auristatin E and derivatives thereof. AFP, AEB, AEVB, MMAF, and MMAE are examples of auristatnica that can be used in this document.

VII. Pharmaceutical formulations

Pharmaceutical formulations of therapeutic ADCs are usually prepared for parenteral administration, for example, in intravenous packaging, intratumoral injection as a pharmaceutically acceptable parenteral vehicle and in an injectable unit dosage form. A conjugate of an antibody to a drug (ADC) having the desired degree of purity is optionally mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.), In form of a lyophilized formulation or an aqueous solution.

Acceptable diluents, carriers, excipients and stabilizers are non-toxic to the recipients of the biological source at the doses and concentrations employed, and include plugs such as phosphate, citrate and other organic acids; antioxidants that include ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, benzyl or butyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including guar gum and dextrins; sugars such as glucose, mannose, sucrose, mannitol, trehalose or sorbitol; chelating agents such as EDTA; salt-forming counterions such as partner; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN ™, Lu Ro NICS ™ or polyethylene glycol (PEG).

VIII. Deglycosylation

The optional stage of deglycosylation is used to reduce the heterogeneity that results from the variable extension of sialic acid and galactose residues in the N-glycans. The heterogeneity of the N-terminus of glycans can be reduced with an enzymatic treatment with exoglycosidase (for example, sialidase and beta-galactosidase, respectively) or by treatment with an endoglycosidase with enzymes that remove N-glycan from the remainder of busy Asn (e.g. , PNGase-F) or enzymes that cleave between the 1st and 2nd residue of N-acetylglucosamine in the N-glycans (for example, Endo-F1, F2 or F3).

In one aspect, the simplest and most effective way to reduce the heterogeneity of N-glycans is to digest with PNGase-F. The deglycosylation step is optional since the quantification is still possible leaving the glycans intact. However, the MS spectrum is unnecessarily complicated and a high-end mass spectrometer is necessary for reproducible quantization.

IX. Chromatography

The removal of non-volatile salts and ionic and non-ionic surfactants from the protein samples occurs before the mass measurement. In one embodiment, the ADC is introduced into the mass spectrometer and is free of non-volatile salts. In another embodiment, the ADC is introduced into the mass spectrometer and is substantially free of non-volatile salts. In another embodiment, the ADC is introduced into the mass spectrometer and is free from nonvolatile salts, with the exception of surfactants, for example, techniques desalting D ^c joined cistelna must maintain intact native protein structure while the non-ionic salts and the surfactants are also eliminated. The types of chromatographic column that can desalt and preserve the native structure in MS-compatible buffer systems include exclusion by size (SE) and HIC columns. It is envisaged that columns which are not marked as SEC columns can be used, for example, HIC columns, in a SEC type mode. The columns are run, for example, in isocratic ammonium acetate buffer. The column operates in such a way as to allow the protein to enter the pores of the chromatographic medium while allowing the non-volatile salts to enter the pores of the chromatographic medium, thus causing the result. that the protein elutes first in the cell conditions and the salts elute later. The migration of non-volatile salts is delayed with respect to the migration of the protein.

The SEC columns employ a chromatographic medium that is usually composed of a stationary silica phase, polystyrene-divinylbenzene or polyhydroxyethyl-aspartamide in the form of parrculates ranging in size from 1.7 to 20 micrometres in diameter with pore sizes ranging from 60 to 2000 angstroms. In one aspect, the pores are 60, 80, 100, 120, 150, 200, 300, 500, 1000, 2000, or 4000 A. In one aspect, any material packaged in a SEC column and column sizes can be used. they can be from 0.1 to 7.8 mm in internal diameter and 50 to 600 mm in length. The column sizes can be 0.1, 0.15, 0.3, 0.5, 1, 2.0, 2.1, 3.0, 4.0, 4.6 or 7.8 mm. The column lengths can be 50, 100, 150, 200, 250, 300 mm.

This class of columns eliminates the non-volatile salts and surfactants of proteins due to the immense difference in size of one with respect to the other. Specifically, the small molecule salts and interaction surfactants (for example, enter the pores of the chromatographic particles that cause these species to be delayed in their passage through the column while the proteins of sufficient size do not interact With the porous structure of the particles and thus the non-volatile salts and surfactants are eluted well Another aspect of a desalination system is the choice of a buffer salt and the pH and the concentration of the salt. volatil to avoid crystallization and corrosion of the MS inlet The commonly used MS buffer volatile salts are ammonic / formalic acid and ammonium acetate / acetic acid (eg, acid / base pairs). The columns can be washed with ammonium acetate at pH 7 and titrated to a pH of 6.5 with acetic acid In one aspect, the salt is ammonium acetate.

The concentration of the salts is between 50 and 400 mM. In one aspect, the salt concentration is 200 mM. In another aspect, the salt concentration is 250 mM. The pH of the buffer is between 5.0 and 7.0. In one aspect, the pH is from 5.5 to 7.0. In another aspect, the pH is 6.5. In still another aspect, the pH is 7.0. One skilled in the art understands that the pH range is expected to result in useful mass data (for example, useful mass data at a pH of 7.5 or 8.0 can be obtained).

The consequent denaturation and fragmentation of an ADC can be avoided if analysis occurs in the absence of organic solvents and acidic pairing reagents. In one aspect, 200 mM of ammonium acetate at a pH of 6.5, used in conjunction with a polyhydroxyethyl-A column provides for the desalination of a deglycosylated IgG1 mAb.

X. Mass spectrometry

In a MS procedure, a sample is loaded into the MS instrument. The sample contains a conjugate compound of a protein with an agent. The sample is ionized with an acid or base for negative or positive ionization, respectively. The sample enters the course as a fine pulverization or droplets. The sample containing droplets is desolvated (evaporated) with a heated drying gas. The size of the droplets decreases to the point where the positively charged protein molecules cause the droplets to explode into smaller droplets due to electrostatic repulsions. This procedure is repeated many times in a second division until it has true gas phase protein ions that later enter the mass spectrometer for the determination of the mass. This process is usually referred to as electrospray ionization (ESI). The mass to charge ratio (m / z) of the molecular ions are determined and these raw data are further processed (broken down) with the vendor's specific software to result in a zero charge molecular mass of the analyte of interest . MS is used to study pharmacokinetics, identify a protein or small unknown molecules and characterize the post-translational modifications and degradations that occur in proteins. The typical ESI instrumentation includes Xevo Q-TOF water, Agilent 6510 Q-TOF.

The spectrometric data are used to confirm the primary structure of the proteins. The confirmation of the primary structure is obtained when the experimentally measured mass of a protein coincides with the theoretical mass, which is determined from the expected amino acid sequence, with a determined error threshold. Measurement of intact mass of the recombinant mAbs can be carried out using mass spectrometers with time of flight available in the market. Typical desalting processes using denaturing solvent systems often result in experimental mass data for mAbs that are 50 ppm (approximately 7.5 Da) of the theoretical value. In other aspects, the mass data is approximately 25 Da. Native desalination of mAb and ADC using SEC-MS in an ammonium acetate buffer system results in lower ionization efficiencies because fewer basic residues are positively charged in a neutral pH buffer. The raw data of lower intensity can result in a greater deviation of the mass experimentally measured from the theoretical mass. However, the experimental data obtained in ADC desalted using SEC-MS described herein give rise to mass data that are 50 ppm (approximately 7.5 Da) of the theoretical value and always less than 100 ppm. The high degree of mass precision indicates that the ADC is completely desalted since the common salt adducts that can potentially associate with the protein will increase the mass by at least 18 Da (the mass of an ammonium ion). Conventional strategies for measuring mass of ADCs with covalently linked subunits involve desalination by off-line techniques such as centrifugal filtration, single use of penetration and dialysis gel cartridges followed by MS with desalted ADC nano-spray. Incomplete desaturation of the sample is quite common using this strategy and can result in mass data that deviates from the theoretical value by up to 5000 ppm.

With the elution of the protein, for example, an ADC, from the SEC column in a volatile salt, for example, ammonium acetate, the protein is introduced into the mass spectrometer using available ESI sources capable of desolvating and ionizing proteins. in a buffer that is flowing between 1 and 1000 microliters / minute. The flow rates can be determined by the operator and can depend on the column diameter. For example, a column of 0.1 mm could be operated at a flow rate of 1 microliter / min and a column of 7.8 mm could be operated at around 1000 microliters / min. This flow allows a determination of the mass much faster. Capillary tension, gas flow velocity (desolvation and cover) and cone voltages should be set to values that they are sufficient to desolvate (for example, evaporate the solvent) the ADC but soft enough to minimize the ongoing fragmentation of the ADC into heavy and light chains bound to a drug. When the folded native structure of the ADC is maintained, then it is evident that the raw data as a load wrap between 4800 and 7000 m / z. If the native structure is not maintained then it is evident that a significant ADC population has a load envelope between 2000 and 4000 m / z. The raw data from the MS measurement of the ADC is converted to a zero-charge mass spectrum using a breakdown software (for example, Agilent MassHunter maximum entropla breakdown) and the abundances of each drug species are quantified by dividing the abundance of ions of a particular bound drug species by total ion abundance (sum the abundance of all bound drug species). Mass measurement of intact proteins is carried out on instruments with four times of flight time and tetrapolar flight time (TOF and QTOF, respectively) but it can also be carried out by Fourier transform ionic cyclotron resonance (FT-ICR) and Orbitrap mass spectrometers. In one aspect, this work could be carried out in u mass spectrometers with laser-assisted matrix ionization desorption (MALDI) and high-resolution ion trap mass spectrometers.

In contrast to the present disclosure, previously each fraction previously collected from the chromatography step was individually introduced into the MS. By testing the mass of each constituent fraction of the compounds and conjugates, the sum of the mass of all the collected peaks was calculated to determine the total mass. As set forth herein, the present disclosure provides methods that analyze intact ADCs directly by MS.

XI. Mass spectrometry with electrospray ionization

Masses of relatively high molecular weight compounds such as antibodies can be detected at mass to charge ratios (m / z) which are easily determined by most mass spectrometers (typical m / z ranges) up to 2000, up to 3000, up to 8000). ESI-MS electrospray ionization mass spectrometry, in particular, is suitable for charged, polar or basic compounds and for the analysis of multiple labeled compounds with excellent detection limits. The ESI allows ace! the detection and characterization of large biomolecules, such as conjugates of an antibody with a drug with a molecular weight (MW) of 150,000 or more.

XII. Systems

In some embodiments, the present disclosure provides a system configured to carry out a method described herein. In some of these embodiments, the systems include a mass spectrometer.

In certain embodiments, the mass spectrometer is configured to carry out a method described herein and includes a means for detecting a mass of a conjugate of a protein with a non-covalently associated agent. In certain embodiments, the medium includes a mass spectrometer. In some embodiments, the systems further include means for providing a conjugate compound of a protein with a non-denatured agent in a volatile salt and free of a non-volatile salt. In additional embodiments, the systems include means for the introduction of the conjugate compound of a protein with an agent in a mass spectrometer. In still further embodiments, the systems include means for establishing the mass directly from the conjugate compound of a protein with an agent by mass spectrometry.

XIII. Examples

The following examples exemplify the invention and are not intended to limit it.

materials

Conjugates of an antibody with a drug - The recombinant monoclonal antibodies were expressed in CHO cells and purified according to conventional procedures described in Shukia, A et al, (2007) J Chromatogr B Analyt Techno! Biomed Life Sci 848, 28-39. The conjugation of cysteine residues in the mAb with Val-Cit monomethyl auristantin E (vcMMAE) or maleimidocaproyl monomethyl auristatin F (mcMMAF) was carried out according to established procedures described in Sun, M. et al, (2005) Bioconjug Chem 16, 1282-1290. The antibodies were deglycosylated by adding 1 μl of PNGase F (New England Biolabs, Ipswich, MA) per 100 μg of antibody or DNA and incubated at 37 ° C for at least 4 hours.

Example 1

SEC-MS Chromatography - Mabs and ADCs were separated on a polyhydroxyethyl-A (PolyLC, Columbia, MD) column with dimensions of 2.1 x 200 mm and containing particles of 5 micrometres with pores of 300 A. The column it was equilibrated in 200 mM of ammonium acetate, pH 6.5-7.0 for the analysis of non-denaturing masses or 30% of acetonitrile, 0.2% formic acid for denaturing mass analysis (control). The flow rate was maintained at 0.1 ml / min during execution and the mAb or ADC eluted between 3.5 and 4.5 min. The flow and buffer composition was maintained following the elution of the mAb or ADC and the total cycle time was 10 min per run.

Mass Spectrometry - The mass spectrum data for the mAb and ADC were acquired in an Agilent 6510 QTOF (Agilent, Santa Clara, CA) in positive ESI mode in the range of 1000-8000 m / z. The temperature of the drying gas was 350 ° C and a flow rate for the drying gas and a gas pressure of the nebulizer was 12 l / h and 35 psi, respectively. The capillary tension, of the fragmentary and octopolar RF were set at 5000, 450 and 350, respectively. The raw data was converted into a zero load mass spectrum with a maximum entropla breakdown algorithm with the MassHunter workstation software version B.03.01.

Hydrophobic interaction chromatography (HIC) - The ADCs with vcMMAE and mcMMAF were separated by HIC according to the methods described in McDonagh, CF et al, (2006) Protein Eng Des Sel 19, 299-307. The quantification of drug loading species was determined by UV integration at 280 nm. In Figure 6, the HIC data is used as a comparison of the relative percentages of ADC MR0-8 determined from the intact mass data method described in this document.

In addition to Figure 6, Table 1 shows the area of ionic abundance and the values of the UV HIC area by MR, of species loaded with individual drugs.

Table 1

A comparison of the raw DM data obtained from the sample of the desalted control mAb in the presence of 30% acetonitrile, 0.2% formic acid with respect to the ammonia acetate / desalted ammonia sample indicated that the first The condition completely denatured the antibody as evidenced by the charge cover, which was observed between 3000 and 4000 m / z, while the desalted ammonium acetate resulted in a charge cover observed between 4800 and 6800 m / z (Fig. 2). , panels A and B respectively). The apparent ions between 200 and 3500 m / z in panel B is the region in which the denatured antibody will be evident are due to the presence of PNGase F which was used for deglycosylation and the non-ionic detergent Tween-80. The disaggregated mass of the antibody determined by any method was at 10 ppm of the theoretical value (Fig. 2, panels C and D).

The mAb-A mcMMAF conjugate described above was also analyzed using the denaturing and non-denaturing chromatographic desalting methods. Desalting the ADC in the presence of 30% acetonitrile, 0.2% formic acid resulted in a charge envelope between 1500 and 4000 m / z (Fig. 3, panel A). The disaggregated MS was dominated by bound antibody fragments including 1LC with 1 drug, 1LC1HC with 2 drugs, 2HC with two drugs and 1LC2HC with a drug and 2LC with 2 drugs with 2 drugs (Fig. 3, panel C). The control conditions showed no evidence of the presence of intact ADC-A. The same m / z region in the desalted ADC MS in ammonium acetate (Fig. 3, panel B) was also broken down and the observed fragmentation of the ADC was much lower and only 1LC with 1 drug and 1LC1HC with 2 drugs were observed. Only multiple carded ions were observed for ADC-A in the raw data for the ammonium acetate desalted sample and indicated than the area between brackets in Fig. 3, panel B.

Analysis of the mCMMAF and vcMMAE ADCs by the same method of ammonium acetate desalination described herein above resulted in a distribution of species with masses consistent with the mAb with 0-8 drug incorporation. The intact mass spectrum of the ADC-A conjugate with mcMMAF and the parent material (mAb-A) are shown in Fig. 4. The relative ionic intensities of the charged species of an individual drug were quantified by the ionic abundance associated with the mass spectrum broken down. A comparison of the relative amounts of ADC MR 0-8 determined from the intact mass data and an orthogonal method, HIC, are shown in Fig. 6, panel A. Similar results were obtained in the analyzes of the ADC conjugate -B with vcMMAE and the parental material (mAb-B) and are shown in Fig. 5. The comparison of the relative amounts of ADC MR 0-8 were determined from the intact mass data and by HIC are shown in Fig. 6, panel B.

Example 2

Exclusion chromatography by size - ADC samples were analyzed off-line using a dual SEC column. Two columns of 7.8 mm x 30 cm of TSK gel G3000SWXL packaged with particles of 5 pm with pores of 250 A. (Tosoh, JPN) were connected in series. The ADC samples were separated using a mobile phase consisting of 200 mM ammonium acetate, pH 7 with a flow rate of 0.8 ml / min. The absorbance at 280 nm was used for the detection of absorbance due to the background interference of the mobile phase at lower wavelengths.

The effect of the desalting method on the dissociation of ADC subunits was evaluated by collecting the ADC from the desalination column, by SEC column dial. Referring to Figure 7, the comparison of the chromatograms of the SEC column analysis of the desalted ADC and the corresponding starting material indicated that there is no talk of an increase in dissociation as a consequence of the desalination method.

Example 3

Experiment - The PHEA SEC column was equilibrated in ammonium acetate buffer at different salt concentrations ranging from 50 mM to 400 mM. The deglycosylated ADC-B was analyzed by SEC MS operated with the cell phase salts concentrations described above and the relative levels of MR0-MR8 were quantified based on the detailed ionized intensity described previously.

Results - At a salt concentration of 50 mM the levels of MR2 and MR6 appear to be over and underrepresented with respect to the levels observed in all other salt concentrations. This would suggest that the ammonium acetate concentration of the mobile phase of SEC-MS should be above 50 mM and that the concentration of ammonium acetate salt of the mobile phase between 100 and 400 mM does not produce widely disparate results with respect to the relative MR distribution.

Figure 8 illustrates that the concentration change of the ammonium acetate in the mobile phase has no impact on the relative distribution of individual charged drug forms when the salt concentration is between 100 and 400 mM.

Example 4

Experiment - The ADC-C is a molecule of IgG1 conjugated with mc-MMAF. The different forms of (loaded drugs were separated and purified by HIC chromatography (Fig. 9A) .The purified fractions, as well as the starting material of the ADC-C (before the separation with HIC) were subsequently evaluated by SEC-. MS as previously described (Fig. 9B).

Results - The results of the SEC-MS are consistent with the orthogonal data (LCMS separation of the reduced light and heavy chains) showed that the AF peaks correspond to different forms of drugs loaded with ADC-C (Fig. 9B). The peaks of the HIC Cy D have the same nominal mass and the most is consistent with MR4 species. The breakdown of raw data in the low m / z range of 1500-4000 (Fig. 10) indicates that peak C is composed primarily of MR4 with drugs incorporated into the antibody Fab as evidenced by the LC number and HC joined to drugs with 2 drugs. A similar analysis HIC peak D indicates that this compound MR4 primarily with drugs incorporated into the antibody hinge as evidenced by C-LC covalent ^h 2 drugs. Based on the information obtained from the analysis of the dissociated antibody chains, it is possible to determine the identities of the positional isomers of MR4 separated by HIC in the peaks C and D.

Figure 9 shows the results of a characterization of the separation of an ADC with mcMMAF by HIC (panel A), followed by the analysis of the fractions collected by SEC-MS (panel B). In Figure 20, an analysis of the individual fractions of the HIC separation shows that the position of the drug binding in the polypeptide chains of ADC can be assessed by the mass of the dissociated fragments using SEC-MS.

Example 5

Micro flow SEC-MS chromatography - The ADCs were separated using a column of polyhydroxyethyl-A (PolyLC, Columbia, MD) with dimensions of 0.3 x 150 mm, containing particles of 5 micrometers with pores of 300 A. The column it was equilibrated with 200 mM ammonium acetate, pH 6.5-7.0 for the non-denaturing mass analysis. The amount of sample loading in the column varied from 2.5 pg to 50 ng. Using an ESI microfluid sprayer device and a positive feedback microflow controller, the flow rate was maintained at 1.0 pl / min for 25 min in a gradient of 200 mM isocratic ammonium acetate, with an ADC elution observed between 15.5 and 19.5 minutes.

Mass spectrometry - The mass spectral data for ADC was acquired using an Agilent 6510 QTOF (Agilent, Santa Clara, CA) in an ESI in positive mode between 1000-8000 m / z. The drying gas temperature was 350 ° C with drying gas and nebulizer pressures set at 5.0 l / h and 15 psi, respectively. All ^other Ms ^{adjustments were identical to analytical-scale experiments except those optimized for the} microflow / microESI ^interface including capillary tension and separator voltages that were set at 3000 and 300 volts respectively. The raw data was converted to neutral mass units using a maximum entropy breakdown algorithm with the MassHunter workstation software version B.03.01.

While the smaller flow rate delayed the elution time in comparison with the analytical method, the detection limit increased drastically, in the four loaded drug species being able to visualize up to 50 ng of deglycosylated ADC in the column. As an example, for the injection of 500 ng of ADC (See Figure 11) the MRD is comparable to the observed SEC method and HIC analysis on an analytical scale. The observed mass / mass error accuracy is also substantially below the fractional specifications.

Example 6

Plasma Stability / Affinity Purification of ADC - Plasma from female Sprague Dawley rats filtered with K2EDTA was incubated with MabSelect to deplete the endogenous IgG. The ADC was added in the IgG-deficient rat plasma at 1 mg / ml, aliquots were made and stored at 37 ° C. The aliquots were removed from storage at 37 ° C and stored at -80 ° C at the specified time points (0, 6 hours, 1 day, 2 days, 4 days, 7 days) until analysis. 500 pl of the 1 mg / ml ADC was then added to the MabSelect, washed in 1 x PBS pH 7.4 three times, eluted in 100 μl of IgG elution buffer, and neutralized in 1 M Tris pH 8 (1:10 v / v).

Micro-flow chromatography SEC-MS - ADCs were separated using a column of polyhydroxyethyl-A (PolyLC, Columbia, MD) with dimensions of 0.3 x 150 mm, containing particles of 5 micrometres with pores of 300 A. The column was balanced with 200 mM ammonium acetate, pH 6.5-7.0 for non-denaturing mass analysis. Using a LC microfluid controller device with positive feedback and a microfluid sprayer in ESI, the flow rate was maintained at 1.0 pl / ml during the 85 minutes gradient of isocratic 200 mM ammonium acetate, with an elution observed between 15.5 and 19.5 minutes. Approximately 1 Hg of ADC was injected for each acquisition.

Mass spectrometry - The mass spectral data for ADC was acquired using an Agilent 6510 QTOF (Agilent, Santa Clara, CA) in an ESI in positive mode between 1000-8000 m / z. The drying gas temperature was 350 ° C with drying gas and nebulizer pressures set at 5.0 l / h and 15 psi, respectively. All ^other Ms ^{adjustments were identical to analytical-scale experiments except those optimized for the} microflow / microESI ^interface including capillary tension and separator voltages that were set at 3000 and 300 volts respectively. The raw data was converted to neutral mass units using a maximum entropy breakdown algorithm with the MassHunter workstation software version B.03.01.

The rebalancing of the column was extended to one hour after each ADC injection purified from the plasma to reduce the interference of any biological contaminant. This increased the signal regarding the noise and the resolution of the ADC peaks. The disaggregated data for the zero time point is as expected, only with the observed charged pair species. As incubation times increase, charged odd species appear and increase in abundance over time (see Figure 12). Although the ADC peaks at the start of the incubation were as expected, the tails of the peaks increased over time. This is due to the heterogeneity of the total sample. Specifically, each loaded drug site is in competition with an addition of reverse Michal or maleimide ring opening. This has been confirmed using a reduced mass analysis. The decrease in MRD observed over time reflects the drug loss in rat plasma deficient in IgG at 37 ° C.

When there is a conflict between the present application and a reference provided in this document, the present application will dominate.

Claims (14)

A method for the detection or determination of a mass of a conjugate compound of an antibody and a drug, comprising: (a) providing a conjugate compound of an antibody with a drug in a volatile salt and free of a non-volatile salt, wherein the conjugate compound of an antibody with a drug has from one to eight drug moieties conjugated to the disulfides reduced intercalans, is non-covalently associated and non-denatured, and maintains its bivalent antibody structure intact. (b) introducing the conjugate compound of an antibody with a drug of step a) into a mass spectrometer; and subsequently (c) directly establishing the mass of the intact bivalent structure of the conjugate compound of an antibody with a drug by mass spectrometry. 2. The method of claim 1, wherein the conjugate compound of an antibody with a drug is present in a distribution of conjugate compounds of an antibody with a drug in a matrix; the method further comprising the application of a separation medium under non-denaturing conditions to effect the separation of the conjugate compounds of an antibody with a drug and the matrix, wherein the conjugate compounds of an antibody with a separate drug are substantially associated not covalently and not denatured, wherein the matrix optionally comprises a non-volatile salt, a surfactant, a buffer or a formulation, and wherein the separation medium optionally comprises a column of HIC or SEC executed under non-denaturing conditions. 3. The method of claim 2 further comprising the quantification by ionic intensity broken down from the relative distribution of the conjugate compounds of an antibody with a separate drug. 4. The method of claim 1 wherein the volatile salt comprises ammonium formate, ammonium acetate or ammonium carbonate. The method of claim 2, wherein the non-denaturing conditions comprise a volatile SEC buffer compatible with mass spectrometry or a temperature not higher than 50 ° C. 6. The method of claim 2, wherein the separation medium comprises an exclusion chromatography column by size (SEC), wherein the SEC column is a polystyrene-divinylbenzene or polyhydroxyethyl aspartamide column. The method of claims 1 or 2, wherein the drug is a maytansinoid, an auristatin, monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). The method of claim 1, wherein the mass spectrometry is carried out by ESI-MS. 9. The method of claim 1, wherein the concentration of the volatile salt ranges from 50 to 400 mM, and optionally wherein the pH provided by the volatile salt is from 5.5 to 7.0. The method of claim 2, wherein the conjugate compounds of an antibody with a drug are immediately introduced into the mass spectrometer, or wherein the conjugate compounds of an antibody with a drug are continuously introduced into the Mass spectrometer. The method of claim 6, wherein the medium of the SEC comprises a polyhydroxyethyl-A column optionally wherein the size of the SEC column is from 0.1 to 7.8 mm internal diameter and 100 at 300 mm in length. The method of claim 1, further comprising the step of treating the conjugate compound of an antibody with a drug with a deglycosylating reagent, wherein the deglycosylating reagent comprises PNGase F or wherein the deglycosylating agent optionally comprises an enzymatic treatment with an exoglycosidase, a treatment with an endoglycosidase or an enzymatic treatment which cleaves between the 1st and 2nd residues of N-acetylglucosamine of the N-glycans, wherein the enzymatic treatment with an exoglycosidase optionally comprises a sialidase or a beta-galactosidase, or wherein the enzymatic treatment that cleaves between the 1st and 2nd N-acetylglucosamine residues of the N-glycans comprises Endo-F1, F2 or F3. The method of claim 1, wherein the antibody of the conjugate of an antibody to a drug is a humanized antibody. The method of claim 1, wherein the antibody of the conjugate of an antibody to a drug is selected from the group consisting of an anti-CD30, anti-CD40, anti-CD19, anti-CD33 and anti-anti-CD33 antibody. -CD70.